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Abstract:

Disclosed are methods of predicting responsiveness of a cancer cell to a
tyrosine kinase inhibitor, and methods of predicting the risk of
progression of a cancer cell to a more aggressive form. Also provided are
methods of reducing proliferation or promoting differentiation of a
cancer cell having reduced level of Numb or increased level of Msi.
Further disclosed are methods of treating a mammalian subject having
cancer and methods of assessing an agent for chemotherapeutic potential.

Claims:

1. A method of predicting responsiveness of a cancer cell in a sample to
a tyrosine kinase inhibitor, comprising evaluating the level of Numb
expression in the cancer cell and using the level of Numb expression to
predict the responsiveness of the cancer cell to the tyrosine kinase
inhibitor.

2. The method of claim 1, wherein the cancer cell is a lymphoma, a
lymphoid leukemia cell, a myeloid leukemia cell, a glioblastoma cell, or
a breast cancer cell.

3. The method of claim 1, wherein reduced Numb expression relative to a
control is predictive of non-responsiveness to the tyrosine kinase
inhibitor.

4. The method of claim 1, wherein the tyrosine kinase inhibitor is
selected from the group consisting of imatinib mesylate, nilotinib, and
dasatinib.

5. A method of predicting the risk of progression of a cancer cell in a
sample to a more aggressive form, comprising evaluating the level of Numb
expression in the cancer cell and using the level of Numb expression to
predict the risk of progression to a more aggressive form.

6. The method of claim 5, wherein a reduced level of Numb expression
relative to a control is indicative of an increased risk of progression.

7. The method of claim 5, wherein the cancer cell is a lymphoma, a
lymphoid leukemia cell, a myeloid leukemia cell, a glioblastoma cell, or
a breast cancer cell.

8-10. (canceled)

11. A method of reducing proliferation or promoting differentiation of a
cancer cell having reduced expression of Numb protein relative to a
control, comprising contacting the cell with an agent capable of
increasing the level of Numb protein in the cell.

22. The method of claim 16, the agent is capable of decreasing MSI1,
MSI2, or both.

23-34. (canceled)

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent
Application No. 61/178,370, filed May 14, 2009, and U.S. Provisional
Patent Application No. 61/332,943, filed May 10, 2010, and both are
incorporated by reference herein in their entireties.

BACKGROUND

[0003] The National Cancer Institute estimates that in the United States
one in three people will be afflicted with cancer. Moreover,
approximately 50% to 60% of people with cancer will eventually die from
the disease. Early intervention and targeted therapeutic treatment are
needed to increase cancer survival. The present invention relates to
methods for cancer diagnosis and treatment.

[0004] In particular, myeloid leukemia may present as a slow-growing
chronic phase with cells able to undergo differentiation (Chronic Myeloid
Leukemia, CML) or as a more aggressive and fast-growing acute phase with
cells that are unable to differentiate (Acute Myeloid Leukemia, AML).
There is a need in the art for methods of distinguishing the various
stages of myeloid leukemia and predicting responsiveness of leukemias to
types of chemotherapeutics. In addition, there is a need for methods of
screening cancer for chemotherapeutic sensitivity and for developing
novel therapeutics.

SUMMARY

[0005] In one embodiment, methods of predicting responsiveness of a cancer
cell to a tyrosine kinase inhibitor are provided. The methods include
evaluating the level of Numb expression in the cancer cell and then using
the level of expression to predict responsiveness to the inhibitor.

[0006] In another aspect, methods of predicting the risk of progression of
a cancer cell to a more aggressive form are provided. These methods
include evaluating the level of Numb expression in the cancer cell and
using the level of expression to predict the risk of progression to a
more aggressive form of the cancer.

[0007] In yet another aspect, methods of reducing proliferation or
promoting differentiation of a cancer cell having reduced expression of
Numb relative to a control are provided. These methods include contacting
the cell with an agent capable of increasing the level of Numb in the
cell. The increased level of Numb reduces proliferation and promotes
differentiation of the cells.

[0008] In still another aspect, methods of treating a mammalian subject
having a cancer that has a reduced level of Numb are provided. The
methods include administering an agent capable of increasing the level of
Numb to an amount effective to treat the cancer.

[0009] In a further aspect, methods of assessing an agent for
chemotherapeutic potential are provided. The methods include contacting a
cell with the agent and evaluating the level of Numb in the contacted
cell. The level of Numb in the contacted cell is indicative of the
chemotherapeutic potential of the agent.

[0010] In still a further aspect, methods of reducing proliferation or
promoting differentiation of a cancer cell having increased expression of
Msi are provided. The methods include contacting the cell with an agent
capable of decreasing the level of Msi in the cell to reduce
proliferation or increase differentiation.

[0011] In yet another aspect, methods of treating a mammalian subject
having a cancer that has an increased level of Msi expression relative to
a control are provided. The methods include administering an agent
capable of decreasing the level of Msi to the subject in an amount
effective to treat the cancer.

[0012] In a further aspect, methods of assessing an agent for
chemotherapeutic potential are provided. The methods include contacting a
cell with the agent and evaluating the level of Msi in the contacted
cell. The level of Msi in the contacted cell is indicative of the
chemotherapeutic potential of the agent.

[0013] In yet another aspect, methods of predicting the risk of
progression of a cancer cell to a more aggressive form are provided.
These methods include evaluating the level of Msi expression in the
cancer cell and using the level of expression to predict the risk of
progression to a more aggressive form of the cancer.

[0014] Other aspects of the invention will become apparent by
consideration of the detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1A are images of AML and CML cells stained with anti-Numb
antibody (red) and DAPI (green). FIG. 1B is a graph of fluorescence
intensity of Numb staining for AML and CML cells. FIG. 1C is an image of
a Western Blot showing detection of Numb expression in CML and AML cells.
FIG. 1D is a graph of relative gene expression of Numb as analyzed by
realtime PCR for AML and CML cells.

[0016] FIG. 2A is a schematic diagram of the strategy used to detect Notch
signaling in AML and CML cells. FIG. 2B is a graph of GFP reporter
expression (black line; GFP expression reflects Notch signalling) in CML
cells, and FIG. 2C is a graph of GFP reporter expression in AML cells,
with expression in wild type cells (gray line) used as a control.

[0017] FIG. 3A is a graph of the survival rate of wild type or Rag1-/-
mice that were transplanted cells transduced with BCR-ABL/Vector,
ICN/Vector, or BCR-ABL/ICN. FIG. 3B is a graph of the average percentage
of blasts in leukemias transduced with BCR-ABL/ICN or BCR-ABL/Vector.
FIG. 3C is an image of wright stained cells derived from wild type
BCR-ABL/Vector driven leukemia. FIG. 3D is an image of wright stained
cells derived from wild type BCR-ABL/ICN driven leukemia. FIG. 3E is an
image of wright stained cells derived from Rag1-/- BCR-ABL/Vector driven
leukemia. FIG. 3F is an image of wright stained cells derived from
Rag1-/- BCR-ABL/ICN driven leukemia.

[0018] FIG. 4A is a graph of the survival rate of mice transplanted with
bone marrow cells infected with BCR-ABL+NUP98-HOXA9 and either Vector or
NUMB. FIG. 4B is a graph of a FACS analysis showing the percentage of
differentiated leukemic cells from mice transplanted with
BCR-ABL/NUP98-HOXA9/Vector or BCR-ABL/NUP98-HOXA9/Numb infected cells.
FIG. 4C is a graph of FACS analysis showing the average percentage of
lineage negative cells from control or Numb expressing leukemias. FIG. 4D
is a graph of the survival rate of mice transplanted with cells from
primary transplanted mice. FIG. 4E-J are images of spleen sections from
leukemic Vector infected transplants (E, H), leukemic Numb infected
transplants (F, I), and healthy Numb infected transplants (G, J).

[0019] FIG. 5A is a graph of primary colony number for AML cells infected
with either control Vector-GFP or Numb-GFP. FIG. 5B is a graph of
secondary colony number for AML cells infected with either control
Vector-GFP or Numb-GFP. FIG. 5C is a graph of the survival of mice
transplanted with AML cells infected with either vector or Numb. FIG. 5D
is a graph of a representative example of lineage analysis for leukemic
spleen cells from primary transplanted mice, wherein AML cells were
analyzed for frequency of lineage negative population. FIG. 5E is a graph
of the average percentage of lineage negative population from primary
transplanted mice. FIG. 5F is the cancer stem cell frequency in primary
leukemias. FIG. 5G is a graph of the average stem cell frequency in
primary leukemias. FIG. 5H is a graph of the survival rate of mice
transplanted with cells from primary transplanted mice that were sorted
for donor derived cells. FIG. 5I is an image of leukemic spleen cells
from secondary transplanted mice infected with control Vector-GFP, with
representative myeloblasts indicated by arrows. FIG. 5J is an image of
leukemic spleen cells from secondary transplanted mice infected with
Numb-GFP, with representative differentiated myeloid cells indicated by
arrows.

[0021] FIG. 7A is a schematic of the strategy to detect Notch signaling in
chronic and blast crisis CML. FIG. 7B shows GFP expression in donor
derived cells from chronic CML as analyzed by FACS for TNR reporter
activity. FIG. 7C shows GFP expression in donor derived cells from blast
crisis CML as analyzed by FACS for TNR reporter activity. FIGS. 7D and 7E
show blast crisis CML cells from TNR mice that were cytospun and
immunostained with anti-cleaved Notch1 antibody and DAPI.

[0022] FIG. 8A shows survival for blast crisis CML cells that were
infected with either vector control or dominant negative Xenopus
Suppressor of Hairless (dnXSu(H)), sorted, and transplanted into
irradiated recipients. FIG. 8B shows survival for cells from primary
transplanted mice that were sorted for donor-derived cells and serially
transplanted into irradiated recipients. FIG. 8C shows survival for bone
marrow progenitors from control (+/+) mice or conditional Rbpj knockout
(Rbpj-/-) mice that were infected with NUP98-HOXA9 and BCR-ABL and
transplanted into irradiated recipients. FIG. 8D shows survival for cells
from primary transplanted mice that were sorted for donor-derived cells
and serially transplanted into irradiated recipients.

[0023] FIG. 9A shows Lin- cells from Vector- or Numb-expressing
NUP98-HOXA9/BCR-ABL-induced leukemia that were sorted, cytospun, and
immunostained with anti-p53 and DAPI. FIG. 9B shows survival for bone
marrow cells from p53 null mice (p53-/-) that were infected with BCR-ABL
and NUP98-HOXA9 together with either Vector or Numb and transplanted into
irradiated recipients. FIG. 9C shows survival for donor-derived cells
from primary transplants that were re-transplanted and monitored for
secondary disease. FIG. 9D shows the frequency of the Lin- population
from primary and secondary leukemia. FIG. 9E shows the number of colonies
from cells from primary transplants that were sorted and plated in
methylcellulose media. FIG. 9F shows the number of colonies from cells
from primary plating that were harvested and replated for secondary
colonies.

[0024]FIG. 10 is a graph of relative gene expression of Numb from human
CML-MMR, CML-no MMR, and bcCML patients.

[0025] FIG. 11A is a graph of the relative gene expression of Msi2 in AML
and CML cells. FIG. 1B is a graph of the relative gene expression of Msi2
in AML sorted into Lin- or Lin+ fractions based on the expression of
lineage markers.

[0026] FIG. 12A shows Musashi (Msi) expression in whole bone marrow (WBM),
KLS cells, chronic and blast crisis CML, olfactory bulb (OB), -reverse
transcriptase (-RT in OB), and water. FIG. 12B is a graph of Msi2
expression as determined by Realtime PCR in KLS cells and Lin.sup.+
cells. FIG. 12C is a graph of Msi2 expression as determined by Realtime
PCR in blast crisis phase and chronic phase cells. FIG. 12D is a graph of
Msi2 expression as determined by Realtime PCR in lin.sup.- chronic and
blast crisis phase cells relative to normal KLS and lin.sup.+ cells. FIG.
12E is a graph of Msi2 expression as determined by Realtime PCR in
lin.sup.- or lin.sup.+ blast crisis CML cells. FIG. 12F are control
vector- or Msi2-expressing CML cells that were stained with anti-Numb
antibody and DAPI. FIG. 12G is a graph of fluorescence intensity
quantified for the cells in 12F. FIG. 12H is a graph of Msi2 expression
in KLS cells transduced with either control vector or NUP98-HOXA9
retrovirus along with BCR-ABL. FIG. 12I is a schematic diagram of the
murine Msi2 gene structure. FIG. 12J shows ChIP performed either with IgG
control or anti-HoxA9 antibody for Flt3. FIG. 12K shows ChIP performed
either with IgG control or anti-HoxA9 antibody for Msi2 -5.7 kb region.
FIG. 12L shows ChIP performed either with IgG control or anti-HoxA9
antibody for Msi2 +110 kb region. FIG. 12M shows β-galactosidase
reporter activity for KLS cells from Msi2 genetrap reporter mice that
were transduced with BCR-ABL with either control vector or NUP98-HOXA9.

[0027] FIG. 13A is a schematic illustration of a Msi2 mutant allele
generated by an insertion of a genetrap vector pU-21. FIG. 13B is a graph
of Msi2 expression in whole bone marrow cells from control (+/+) and
homozygotes for the genetrap allele (Gt/Gt) as analyzed by Realtime
RT-PCR.

[0028] FIG. 14A is the frequency of KLS cells in mice of indicated
genotypes (+/+, +/Gt, Gt/Gt). FIG. 14B shows is a survival curve of mice
transplanted with BCR-ABL and NUP98-HOXA9 infected +/+ or Gt/Gt KLS
cells. FIG. 14C is a graph of colony numbers for blast crisis CML cells
transduced with control shRNA (shLuc) or Msi2 shRNA (shMsi). FIG. 14D is
a survival curve of mice transplanted with established blast crisis CML
cells infected with control shLuc or shMsi. FIG. 14E are Wright's stain
of leukemic cells from mice transplanted with control shLuc or shMsi
infected blast crisis CML. FIG. 14F is a survival curve of mice
transplanted with Lin.sup.- cells from primary shRNA expressing
leukemias.

[0029] FIG. 15A is a graph of Msi2 expression in Lin- cells from
NUP98-HOXA9/BCR-ABL-induced blast crisis leukemias that were infected
with either shLuc or shMsi. FIG. 15B is a graph of Msi2 expression after
Msi2 knockdown by an independent lentiviral shRNA construct, shMsi-2.
FIG. 15C is a graph of the number of colonies formed with an independent
shRNA construct, shMsi-2. FIG. 15D is a graph of Msi2 expression in
samples expressing the indicated constructs. FIG. 15E is a graph of the
colony-forming ability of the samples in 15D.

[0030]FIG. 16 is a graph of survival of mice transplanted with AML cells
transduced with Msi2 shRNA or with a control shRNA.

[0032] FIG. 18A is a graph of MSI2 expression in chronic and blast crisis
CML patient samples from the Korean Leukemia Bank. FIG. 18B is a graph of
MSI2 expression in chronic and blast crisis CML patient samples from the
Hammersmith MRD Lab Sample Archive. FIG. 18C is a microarray analysis of
expression of MSI2 in bone marrow and peripheral blood samples. FIG. 18D
is a microarray analysis of expression of NUMB in bone marrow and
peripheral blood samples. FIG. 18E is a microarray analysis of expression
of HOXA9 in bone marrow and peripheral blood samples. FIG. 18F is a
microarray analysis of expression of HES1 in bone marrow and peripheral
blood samples. FIG. 18G is a schematic diagram of a proposed model for
the role of Musashi and Numb in CML progression.

[0033] FIG. 19A is a graph of HES1 expression during CML progression
within a single patient. FIG. 19B is a graph of TRIB2 expression in
chronic and blast crisis phase cells from CML patient samples in
individual samples. FIG. 19B is a graph of average TRIB2 expression
levels in chronic and blast crisis phase cells from CML patient samples.

DETAILED DESCRIPTION

[0034] As described in the Examples, it was discovered that CML cells in
general expressed significantly higher levels of Numb as compared to AML
cells. As used herein, the aggressive from of CML is referred to either
as blast crisis CML (bcCML) or acute phase leukemia (AML). Interestingly,
a subpopulation of CML cells expressing lower levels of Numb was
identified. Surprisingly, the CML cells expressing lower levels of Numb
correspond to CML cells unresponsive to treatment with imatinib, a
tyrosine kinase inhibitor. Thus it was discovered that the level of Numb
expression in a cancer cell can be used to predict responsiveness of CML
cells to chemotherapeutics. Further, it is appreciated that therapeutics
directed at increasing Numb expression within the cancer cells may
decrease the aggressiveness of the cancer and increase responsiveness to
other chemotherapeutics.

[0035] In addition, it was also discovered that Musashi, namely Msi2,
expression downregulates Numb expression. Thus, Musashi expression may
also be predictive of chemotherapeutic responsiveness. Additionally, it
was discovered that reducing expression of Musashi in the cancer cells
results in increased Numb expression, reduces the aggressiveness of the
cancer and increases the responsiveness of the cancer to
chemotherapeutics.

[0036] Methods of predicting responsiveness of a cancer cell to a tyrosine
kinase inhibitor are provided herein. The method includes evaluating the
level of Numb expression in the cancer cell and then using the level of
Numb expression in the cancer cell to predict the responsiveness of the
cancer cell to the tyrosine kinase inhibitor. As shown in Example 6, CML
cells that do not respond to treatment with the tyrosine kinase inhibitor
imatinib were found to have reduced expression of Numb. Accordingly,
reduced Numb expression relative to a control is predictive of
non-responsiveness to tyrosine kinase inhibitors, including imatinib.

[0037] The level of Numb expression in the cancer cell may be evaluated by
a variety of techniques, as will be appreciated by those of skill in the
art. For example, the level of Numb expression may be evaluated at either
the protein or mRNA level using techniques including, but not limited to,
Western blot, ELISA, Northern blot, real time PCR, immunofluorescence, or
FACS analysis. In Example 2, the expression level of Numb was evaluated
by immunofluorescence by visualizing cells stained with a
fluorescently-labeled Numb-specific antibody, Western blot analysis of
Numb protein expression, and RT-PCR of Numb transcripts.

[0038] As stated above, the expression level of Numb may be compared to a
control. A control may include comparison to the level of Numb expression
in a control cell, such as a noncancerous cell or a cancer cell with
known responsiveness to a therapeutic. Alternatively a control may
include an average range of the level of Numb expression from a
population of chemotherapeutic responsive CML cells, non-cancer cells,
chemotherapeutic non-responsive cells, or a combination thereof.
Alternatively, a standard value developed by analyzing the results of a
population of cells with known responsivities to tyrosine kinase
inhibitors may be used. Those skilled in the art will appreciate that a
variety of controls may be used.

[0039] Predicting may include using the information found in the Examples
or generated by another entity to generate predictions. Predictions may
be based on a comparison internal to a single assay or by comparison to a
standard. For example the level of expression of Numb may be used to
predict a cancer's responsiveness to a therapeutic. Predictions may be
generated in relation to a standard or control as discussed above. This
does not mean that the predicted event will occur with 100% certainty.
Predicting and prediction also includes, but is not limited to,
generating a statistically based indication of whether a particular event
will occur, e.g. whether the cancer will be responsive to treatment with
tyrosine kinase inhibitors.

[0040] The cancer cells for use in the methods include, but are not
limited to, cancers cells characterized by a lack of differentiation of
the cell. Examples of this type of cancer cell may include, but are not
limited to, a leukemia cell, a breast cancer cell, a glioblastoma cell,
or a lymphoma cell. The leukemia cell may be myeloproliferative disorder
including but not limited to preleukemia. The leukemia cell may be a
medullablastoma cell. The leukemia cell may be a myeloid or a lymphoid
leukemia cell. Suitably, the cancer cell may be a myelogenous leukemia
cell. Suitably, the cancer cell may be a CML or AML cell.

[0041] Tyrosine kinase inhibitors encompass agents that inhibit the
activity of one or more tyrosine kinases. For example, a tyrosine kinase
inhibitor may indirectly or directly bind and inhibit the activity of the
tyrosine kinase, including binding activity or catalytic activity. A
tyrosine kinase inhibitor may prevent expression of the tyrosine kinase,
or inhibit the ability of the tyrosine kinase to mediate phosphorylation
of its target. Examples of tyrosine kinase inhibitors include, but are
not limited to, imatinib mesylate, axitinib, bosutinib, cediranib,
dasatinib, erlotinib, gefitinib, lapatinib, lestaurtinib, nilotinib,
semaxanib, sunitinib, vandetanib, and vatalanib.

[0042] The level of Numb protein correlates to the resistance or
sensitivity of a cancer cell to treatment with a tyrosine kinase
inhibitor. For example, reduced expression of Numb relative to a control
cell that is responsive to a tyrosine kinase inhibitor is indicative of
non-responsiveness. Increased expression of Numb relative to an AML cell
is indicative of responsiveness of the cell to tyrosine kinase
inhibitors. In FIG. 7, non-responsive CML cells expressed Numb at a level
20% of that of responsive CML cells. Cells expressing less than about
50%, about 40%, about 30%, about 20%, about 10%, about 5%, or about 1% as
much Numb as control cells responsive to treatment are predicted to be
non-responsive to treatment with tyrosine kinase inhibitors. Conversely,
cells expressing more than about 50%, 60%, 70%, 80%, 90%, or 100% as much
Numb as control cells responsive to treatment are predicted to be
responsive to treatment with tyrosine kinase inhibitors. It is envisioned
that cells having more than 2, 3, 4, 5, 6, 8, or 10 fold higher Numb
expression than control cells non-responsive to treatment may be
responsive to treatment with tyrosine kinase inhibitors, and cells
expressing less than 125%, 100%, or 90% as much Numb as control cells
non-responsive to treatment are predicted to be non-responsive to
treatment with tyrosine kinase inhibitors.

[0043] In another embodiment, methods of predicting the risk of
progression of a cancer cell to a more aggressive form are provided. The
methods include evaluating the level of Numb expression in a cancer cell
and using the level of expression to predict the risk of progression to a
more aggressive form. Suitably, a reduced level of Numb expression
relative to a control CML cell is indicative of an increased risk of
progression to a more aggressive form. Similarly, increased expression of
Numb correlates with less aggressive cancers. As shown in Examples 2-6,
CML cells expressed significantly higher levels of Numb compared to AML
cells, and Numb expression appears to be reduced with progression from
chronic disease to acute disease. For example, reduced expression of Numb
relative to a control CML cell is indicative of an increased risk of
progression of the cancer cell to a more aggressive form. Increased
expression of Numb relative to a control CML cell is indicative of a
decreased risk of progression of the cancer cell to a more aggressive
form. Thus the risk of progression of the cancer may be evaluated using
methods similar to those described above for predicting responsiveness to
a therapeutic.

[0044] In another embodiment, methods of reducing proliferation or
promoting differentiation of a cancer cell having reduced expression of
Numb protein relative to a control are provided. The cancer cells are
contacted with an agent capable of increasing the level of Numb protein
in a cell. In this embodiment, a control includes a control cell, such as
a non-cancer cell of the same type as the cancer cell or a standard based
on such a control cell.

[0045] Agents capable of increasing the level of Numb protein include any
agent capable of increasing Numb protein or Numb mRNA levels. In one
embodiment, the agent may comprise the Numb protein itself. For example,
the agent may include exogenously expressed and isolated Numb protein
capable of being delivered to the cells. The Numb protein may be
delivered to cells by a variety of methods, including fusion to Tat or
VP16 or via a delivery vehicle, such as a liposome, all of which allow
delivery of protein based agents across the cellular membrane. Those of
skill in the art will appreciate that other delivery mechanisms for
proteins may be used. Alternatively, Numb mRNA expression may be enhanced
relative to control cells by contact with the agent. For example, the
agent capable of increasing the level of natively expressed Numb protein
may include a gene expression activator or de-repressor. The agent
capable of increasing the level of Numb protein may also include agents
that bind to Numb directly or indirectly and increase the effective level
of Numb, for example, by enhancing the binding or other activity of Numb.

[0046] In another embodiment, the agent capable of increasing the level of
Numb protein may comprise a polynucleotide encoding the Numb protein or a
polypeptide having at least 95% amino acid identity to Numb and having
Numb activity. There are several isoforms of the Numb protein, including,
for example, SEQ ID NOs: 12, 14, 16, 18, 20, 22, and 24, each of which
may be encoded by more than one polynucleotide sequence, including, for
example, SEQ ID NOs: 11, 13, 15, 17, 19, 21, and 23 (see polynucleotide
and amino acid sequences appended to this application in a sequence
listing, which is incorporated herein by reference in its entirety).
Envisioned are polynucleotide sequences encoding Numb, a polypeptide
sequence encoding Numb, or a polypeptide having at least 95% amino acid
identity to Numb and having Numb activity. Those skilled in the art will
appreciate that a Numb polynucleotide may be delivered to cells in a
variety of ways, including, but not limited to, transfection,
transduction, transformation, via a vector such as a viral vector or
expression vector, or via a liposome. Suitably the polynucleotide
encoding the Numb protein is operably connected to a promoter such that
the Numb protein is expressed in the cancer cells. As shown in Example 4,
expressing Numb from a vector in AML cells may increase the level of Numb
and promote differentiation (FIG. 4B). It is further shown in the
Examples that leukemias developed in the presence of Numb may be
significantly more differentiated and less aggressive. Suitably, the
agent has a direct effect on Numb protein or mRNA expression. In another
embodiment, the agent capable of increasing the level of Numb protein may
comprise an agent capable of decreasing Msi, as described further below.

[0047] In another embodiment, methods of treating a mammalian subject
having a cancer with a reduced level of Numb protein as compared to a
control are provided. The method may comprise administering to the
subject an agent, such as those described above, capable of increasing
the level of Numb protein in an amount effective to treat cancer. As will
be appreciated by those of skill in the art, an agent may be administered
by various methods including sublingually, orally, enterally,
parenterally, topically, systemically or injected intravascularly or
intraarterially, cutaneously, or peritoneally. Treating cancer includes,
but is not limited to, reducing the number of cancer cells in the
subject, reducing progression of a cancer cell from a chronic to a more
aggressive form, reducing proliferation of cancer cells, killing of
cancer cells, or reducing metastasis of cancer cells.

[0048] In another embodiment, methods of assessing the chemotherapeutic
potential of an agent are provided. The methods may include contacting a
cell with the agent and then evaluating the level of Numb in the
contacted cell. As demonstrated in the Examples, the level of Numb in the
contacted cell is indicative of the therapeutic potential of the agent.
Specifically, increased expression of Numb in cells contacted with the
agent is indicative of an agent with therapeutic potential. Conversely, a
lack of any change or a reduction in Numb expression after contact with
the agent as compared to the level of Numb expression prior to contact
with the agent is indicative of a lack of therapeutic potential of the
agent. In FIG. 10, CML cells responsive to imatinib were shown to express
relatively high levels of Numb protein while those resistant to imatinib
were shown to express relatively low levels of Numb.

[0049] Chemotherapeutic potential of an agent is the assessment of the
agent's capability to act as a chemotherapeutic. Chemotherapeutic
potential may include a prediction of the agent's capability to kill at
least one cancer cell, to reduce the growth rate or proliferation rate of
a cancer cell, to reduce the number of cancer cells in an individual, or
to reduce progression of a cancer cells from a chronic to a more
aggressive form.

[0050] Cells may be contacted with the agent directly or indirectly in
vivo, in vitro, or ex vivo. Contacting encompasses administration to a
cell, tissue, mammal, patient, or human. Further, contacting a cell
includes adding an agent to a cell culture. Other suitable methods may
include introducing or administering an agent to a cell, tissue, mammal,
or patient using appropriate procedures and routes of administration as
defined above.

[0051] In another embodiment, methods of reducing proliferation or
promoting differentiation of a cancer cell having increased Msi
expression are provided. The methods include contacting the cell with an
agent capable of decreasing Msi to reduce proliferation or increase
differentiation of cells. Decreasing Msi includes reducing the level of
Msi expression at the mRNA or protein level or decreasing the activity of
Msi. As used herein, Msi includes Msi1, Msi2, MSI1, MSI2, or combinations
thereof. Also included are homologs and orthologs of Msi such as human
MSI2. As shown in Example 8, the human ortholog MSI2 has the same pattern
of expression in leukemic cells from chronic and blast-crisis CML as the
expression pattern in a mouse model.

[0052] Levels of Msi correlate to aggressiveness of cancer. Thus, methods
of predicting the risk of progression of a cancer cell to a more
aggressive form are provided. These methods include evaluating the level
of Msi expression in the cancer cell and using the level of expression to
predict the risk of progression to a more aggressive form of the cancer.

[0053] As shown in Example 7, Msi2 contributes to the establishment and
maintenance of AML from CML. Further shown in Example 7 is that AML cells
require Msi2 during disease maintenance and propagation. FIG. 16 shows
that decreasing the levels of Msi in a cell reduce cancer cell survival.
Reducing the level of Msi in a cancer cell may reduce cancer cell
survival by at least about 5%, at least about 10%, at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least about
60%, or at least about 70%. A reduction in cancer cell survival may be
achieved by reducing Msi expression or activity by at least about 50%,
40%, 30%, 20%, 10%, 5% or less as compared to the Msi expression or
activity prior to treatment with the agent.

[0054] An agent capable of decreasing Msi includes a variety of agents and
molecules capable of decreasing Msi mRNA transcripts or protein levels in
the contacted cell. In one embodiment, the agent capable of decreasing
Msi may comprise an inhibitory RNA. Suitably, the inhibitory RNA may
comprise a shRNA such as SEQ ID NO: 9 as used in Example 7. An inhibitory
RNA may comprise a sequence complementary to a portion of an RNA sequence
encoding Msi. There are several isoforms of the Msi protein, including,
for example, SEQ ID NOs: 26, 28, and 30, each of which may be encoded by
more than one polynucleotide sequence, including, for example, SEQ ID
NOs: 25, 27, and 29 (see polynucleotide and amino acid sequences appended
to this application in a sequence listing, which is incorporated herein
by reference in its entirety). Envisioned are polynucleotide sequences
encoding Msi, a polypeptide sequence encoding Msi, or a polypeptide
having at least 95% amino acid identity to Msi and having Msi activity.
In some embodiments, the agent capable of decreasing Msi may include a
gene expression repressor. In some embodiments, the agent capable of
decreasing Msi may include a small molecule inhibitor. The agent capable
of decreasing Msi may also include agents that bind to Msi directly or
indirectly and decrease the effective level or activity of Msi, for
example, by inhibiting the binding or other activity of Msi. The agent
capable of decreasing Msi may also include agents that decrease Msi1,
Msi2, or both. As shown in Examples 7 and 8, Msi2 expression was found to
be greater in AML than in CML cells and Msi2 expression may be associated
with immature leukemic cells (FIG. 11). Further, Msi2 knockdown with
shRNA resulted in the longer latency of AML development in the mouse
model of AML (FIG. 16).

[0055] In another embodiment, methods of treating a mammalian subject
having a cancer with increased Msi expression relative to a control are
provided. The methods include administering to the subject an effective
amount of an agent capable of decreasing the level of Msi. Modes of
administering an agent are discussed above. Agents suitable for the
methods are similar to those described above that are capable of
decreasing Msi expression or activity.

[0056] In another embodiment, methods of assessing an agent for
chemotherapeutic potential are provided. The methods include contacting a
cell with the agent and evaluating the level of Msi expression or
activity in the contacted cell. The level of Msi expression or activity
may be evaluated using methods similar to those described above. The
level of Msi may be compared to a control, such as cells prior to contact
with the agent, as described above. The level of Msi expression in the
contacted cell may be indicative of the chemotherapeutic potential of the
agent. As shown in Example 7, Msi2 may reduce the level of Numb protein
and, at least in part, may activate a cascade that leads to inhibition of
differentiation and a less aggressive form of cancer. Msi2 may
downregulate Numb and contribute to the establishment and maintenance of
acute myeloid leukemia. As detailed above, Numb expression may be used as
part of a method to assess an agent for chemotherapeutic potential.
Decreased Msi expression in a cancer cell may increase the level of Numb
in the cancer cell and lead to reduced differentiation and a less
aggressive form of cancer. As one example, reduced expression of Msi
relative to an AML cancer cell control upon administration of an agent is
indicative of an agent that is an effective chemotherapeutic agent.

EXAMPLES

Example 1

Materials and Methods

[0057] Mice

[0058] Mouse models of CML were generated by transducing bone marrow stem
and progenitor cells with retroviruses carrying BCR-ABL (chronic phase),
or BCR-ABL and NUP98-HOXA9 (blast crisis phase) and transplanted into
irradiated recipient mice. The development of CML was confirmed by flow
cytometry and histopathology. Transgenic Notch reporter (TNR), Rag1-/-,
and C57BL6/J were used as transplant donors, and C57B16/J CD45.1 and CD1
mice were used as transplant recipients. All mice were 8-12 weeks of age.
Mice were bred and maintained on acidified water after transplantation in
the animal care facility at Duke University Medical Center. All live
animal experiments were performed according to protocols approved by the
Duke University Institutional Animal Care and Use Committee. For Msi2
knockdown experiments, lineage negative blast crisis CML cells were
infected with Msi2 or control Luciferase shRNA retroviral constructs and
leukemia incidence monitored.

[0062] The BCR-ABL polynucleotide was a gift from Warren Pear and Ann
Marie Pendergast and was cloned into a MSCV-IRES-YFP or a MSCV-IRES-CFP
retroviral vector. HOXA9-NUP98-IRES-YFP was a gift from Gary Gilliland
and Craig Jordan and was also cloned into the MSCV-IRES-NGFR vector. Numb
cDNA (p65 isoform, Accession number BC033459, NCBI) was cloned into the
MSCV-IRES-GFP (and hCD2) vectors. ICN1 was cloned into the MSCV-IRES-CFP
or YFP retroviral expression vectors. Virus was produced by triple
transfection of 293T cells with MSCV constructs along with gag-pol and
VSVG constructs. Viral supernatant was collected for three days and
concentrated 100-fold by ultracentrifugation at 50,000×g. For viral
infection, c-Kit+ enriched or KLS cells were cultured overnight in the
presence of X-Vivo15 (BioWhittaker), 50 μM 2-mercaptoethanol, 10%
fetal bovine serum, SCF (100 ng/mL), and Tpo (20 ng/mL). After 12-18 h,
concentrated retroviral supernatant was added to the cells. Cells were
then incubated at 32° C. for 12 h and 37° C. for 36 h.
Infected cells were then sorted based on their GFP, YFP, CFP, NGFR, or
hCD2 expression as appropriate. All cytokines were purchased from R&D
systems.

[0063] In Vitro Methylcellulose Assays

[0064] Lineage negative, NUP98-HOXA9-YFP positive cells from AML were
sorted and infected retrovirally with either Vector-IRES-GFP or
Numb-IRES-GFP. After 48 hours of infection, cells were sorted and
serially plated on complete methylcellulose medium (Methocult GF M3434
from StemCell Technologies).

[0065] In Vivo Leukemia Models

[0066] Bone marrow c-Kit+ or KLS cells from C57BL6/J or TNR mice were
enriched and cultured overnight in X-vivo 15 with 10% FBS, 50 uM
2-mercaptoethanol, 100 ng/mL SCF, and 20 ng/mL TPO in a 96 well U-bottom
plate or 6 well plate. Subsequently, cells were infected with
MSCV-BCR-ABL-IRES-YFP (or CFP) to generate CML, or MSCV-BCR-ABL-IRES-YFP
(or CFP) and MSCV-NUP98-HOXA9-IRES-YFP (or NGFR) to generate AML. Cells
were harvested 48 hours after infection and transplanted retro-orbitally
into groups of 4-7 recipient mice. Recipients were lethally irradiated
(10 Gy) for CML, and sublethally irradiated (7 Gy) for AML. For Numb
overexpression, cells were infected with either MSCV-Numb-IRES-GFP (or
hCD2) or MSCV-IRES-GFP (or hCD2) along with MSCV-BCR-ABL-IRES-YFP (or
CFP) and MSCV-NUP98-HOXA9-IRES-NGFR (or YFP) and 20,000 to 100,000 sorted
infected cells were transplanted per mouse. For secondary
transplantation, cells from primary transplanted mice were sorted for
either MSCV-Numb-IRES-GFP and MSCV-NUP98-HOXA9-YFP or MSCV-IRES-GFP and
MSCV-NUP98-HOXA9-YFP, and 7,000 to 8,000 cells were transplanted per
mouse. For ICN experiments, cells were infected with MSCV-ICN-IRES-CFP
and MSCV-BCR-ABL-GFP, MSCV-ICN-IRES-CFP and MSCV-IRES-GFP, or
MSCV-BCR-ABL-IRES-GFP and MSCV-IRES-CFP, and 10,000 to 20,000 KLS-derived
or 200,000 to 500,000 c-kit+-derived sorted cells were transplanted per
mouse. For the TNR mouse model, cells were infected with
MSCV-BCR-ABL-IRES-YFP (or CFP) to generate CML or MSCV-BCR-ABL-IRES-YFP
(or CFP) and MSCV-NUP98-HOXA9-IRES-NGFR to generate AML. 10,000 to 80,000
unsorted cells were transplanted per mouse. After transplantation,
recipient mice were subsequently maintained on antibiotic water
(sulfamethoxazole and trimethoprim) and evaluated daily for signs of
morbidity, weight loss, failure to thrive, and splenomegaly. Premorbid
animals were sacrificed by CO2 asphyxiation followed by cervical
dislocation. Subsequently relevant tissues were harvested and analyzed by
flow cytometry and histopathology.

[0067] Immunofluorescence Staining

[0068] For immunofluorescence relevant cell populations were sorted,
cytospun, and fixed in 4% paraformaldehyde for 5 minutes. Samples were
then blocked using 20% normal donkey serum in PBS with 0.1% Tween 20, and
stained at 4° C. overnight with an antibody to Numb (1:200)
(ab4147, Abcam) followed by anti-goat Alexa 594 (Molecular probe) and
DAPI. Slides were mounted using fluorescent mounting media (Fluoromount-G
SouthernBiotech) and viewed by confocal microscopy.

[0080] Cells from AML and CML were sorted, cytospun, and immunostained
with anti-Numb antibody. Analysis of cells from fully developed AML and
CML revealed that CML cells expressed significantly higher levels of Numb
compared to AML cells (FIG. 1A, 1B). Data in FIG. 1A is representative of
three independent experiments, and data shown in FIG. 1B is the average
intensity from a representative experiment (p<0.05). This pattern of
expression was also confirmed by western blotting with Numb protein band
shown at approximately 65-70 kDa (FIG. 1C, data is representative of four
independent experiments). As shown in FIG. 1C, the expression of Numb
protein is 2.6 fold higher in CML cells relative to AML cells.

[0081] This pattern of expression was also confirmed by real time PCR
(FIG. 1D). Briefly, AML and CML cells were sorted and RNA was isolated.
The level of Numb was analyzed by realtime PCR (n=7 for CML; n=9 for AML;
p<0.05), and results were normalized to beta-2-microglobulin
expression levels.

[0083] The pattern of Numb and Notch signaling in AML essentially
corresponded to the rise in the frequency of undifferentiated cells and
suggested that the Numb-Notch pathway may play a functional role in
inhibiting differentiation during AML establishment. To test whether
upregulation of Notch signaling could drive the conversion of chronic
leukemia to a more undifferentiated state, the constitutively active
intracellular domain of Notch1 (ICN) was used (Carlesso, N., Aster, J.
C., Sklar, J. & Scadden, D. T. Blood 1999, 93, 838-48, incorporated
herein by reference in its entirety). Not only wild type mice were used
but also Rag1 deficient (Rag1-/-) mice in which lymphoid development is
blocked (Mombaerts, P. et al. Cell 1992, 68, 869-77, incorporated herein
by reference in its entirety). c-kit+ or KLS cells were infected with
BCR-ABL (n=18), ICN/Vector (n=11), or BCR-ABL/ICN (n=16), and
transplanted into irradiated recipient mice. Analysis of survival over a
period of 90 days revealed that activation of Notch signaling decreased
the latency of leukemia driven by BCR-ABL (FIG. 3A). Data shown in FIG.
3A is a combination of 4 independent experiments (BCR-ABL/ICN versus
ICN/Vector, p<0.0001; BCR-ABL/ICN versus BCR-ABL/Vector, p=0.0194).

[0084] The average percentage of blasts in leukemias from BCR-ABL/ICN
versus BCR-ABL/Vector was also compared. As shown in FIG. 3B, a
significantly higher percentage of blasts were found in leukemias from
BCR-ABL+ ICN (p=0.044; error bars show s.e.m.). The analysis included
data from all mice that could be analyzed before succumbing to disease
(for BCR-ABL/ICN n=6 wild type, and n=6 Rag 1-/- out of a total of 16
transplanted; for BCR-ABL/vector n=3 wild type and n=4 Rag-1-/- out of a
total of 18 transplanted). As shown in FIG. 3B, the cooperative effect of
BCR-ABL and ICN led to more undifferentiated leukemias, with control
leukemias containing on average 6% myeloid blasts and BCR-ABL/ICN
leukemias containing on average 35% myeloid blasts. Cells derived from
wild type BCR-ABL/Vector driven leukemias (FIG. 3C), BCR-ABL/ICN driven
leukemias (FIG. 3D), Rag1-/- BCR-ABL/Vector driven leukemias (FIG. 3E),
BCR-ABL/ICN driven leukemias (FIG. 3F) were also compared, based on
morphological analysis after wright staining splenocytes. Differentiated
myeloid cells are indicated by arrowheads, and myeloblasts are indicated
by arrows in FIG. 3 (magnification 100×). Although a majority of
BCR-ABL/ICN leukemias derived from wild type cells were of the myeloid
lineage (66%, 4/6, FIG. 3C, 3D), two displayed elements of lymphocytic
leukemia (33%, 2/6, data not shown). In contrast, all BCR-ABL/ICN
leukemias derived from Rag1-/- mice were of the myeloid lineage (100%,
6/6, FIG. 3E, 3F), allowing a clearer assessment of ICN contribution to
myeloid leukemia progression. Cumulatively the data from both wild type
and Rag1-/- mice suggested that activation of Notch signaling can inhibit
differentiation and thereby drive the conversion of chronic myelogenous
leukemias to a more undifferentiated state and a more aggressive form of
cancer.

Example 4

Numb Induces Differentiation of Undifferentiated Leukemias

[0085] It was tested whether Numb had the ability to convert
undifferentiated leukemias to a more differentiated state, and thus slow
disease progression. Bone marrow c-kit+ cells were infected with BCR-ABL
and NUP98-HOXA9 together with either control vector or Numb. The cells
were transplanted, and survival and leukemia progression was monitored.
83% of BCR-ABL/NUP98-HOXA9/Vector (control) mice and 63% of those
transplanted with BCR-ABL/NUP98-HOXA9/Numb developed leukemia (FIG. 4A;
data shown is from four independent experiments; n=18 for Vector and n=19
for Numb). Cells were also analyzed via FACS for frequency of lineage
negative cells to determine and compare the level of differentiation in
the BCR-ABL+NUP98-HOXA9/Numb and BCR-ABL+NUP98-HOXA9/Vector cells.
Results indicated that the relative survival increase in cells expressing
Numb was consistent with leukemias developed in the presence of Numb that
displayed increased differentiation (FIG. 4B; cells were analyzed for
frequency of lineage negative cells). Specifically, while the frequency
of lineage negative immature cells was on average 47% in control
leukemias, it was reduced to 12.3% in Numb expressing leukemias (FIG. 4C;
p<0.001; error bars shown s.e.m.). This showed that re-expression of
Numb depleted the most immature fraction of AML and converted it to a
more differentiated disease.

[0086] Secondary transplants were carried out to test the ability of
control and Numb expressing AML cells to propagate disease in secondary
recipients. Cells from primary transplanted mice were sorted and
transplanted, and the mice were monitored for secondary disease. As shown
in FIG. 4D (data shown is from two independent experiments; n=14 for
Vector; n=15 for Numb; **p<0.001), control AML cells were able to
propagate disease in nearly all of the mice ( 8/9, 89%), but Numb
expressing AML cells were significantly impaired in their ability to
propagate the disease and lead to a significant decrease in AML incidence
( 2/10, 20%). Importantly, the few leukemias that did develop in the
presence of Numb were less aggressive than control leukemias.

[0087] Hematoxylin and Eosin staining of Spleen sections from leukemic
Vector infected transplants (FIGS. 4E and 4H), leukemic Numb infected
transplants (FIGS. 4F and 4I; disease at 40 days), and healthy Numb
infected transplants (FIG. 4G, 4J; sacrificed at 150 days) were prepared
to analyze the morphology of the cells. In FIG. 4E-4J, cells were stained
with Hematoxylin and Eosin, and magnification is 10× (E-G) or
63× (H-J), with red arrows indicating areas of immature myeloid
cells and black arrows indicating lymphoid follicles. Error bars in all
bar graphs are s.e.m. Data shown is representative of three to four
independent experiments. While spleen sections from control leukemias
displayed extensive myeloid cell infiltration (FIGS. 4E and 4H red
arrows), and few remaining areas of normal lymphoid follicles, spleen
sections from leukemias expressing Numb showed greater preservation of
lymphoid follicles (FIGS. 4F and 4I, black arrows). In addition,
surviving mice transplanted with Numb AML cells that were sacrificed and
analyzed at 150 days displayed normal splenic architecture and had no
indication of leukemogenesis (FIGS. 4G and 4J). These data indicate that
when expressed during disease initiation, Numb can impair both the
incidence and progression of acute myeloid leukemia by inducing
differentiation.

[0088] The ability of Numb to influence AML progression after disease had
been established was also tested. Fully developed AML cells were infected
with either vector or Numb, and colony-formation was assessed in vitro
using a serial replating assay (Zhao, C. et al. Cancer Cell 2007, 12,
528-41; Huntly, B. J. et al. Cancer Cell 2004, 6, 587-96; both
incorporated herein by reference in their entireties). AML cells were
infected with either control Vector-GFP (MSCV-IRES-GFP) or Numb-GFP
(MSCV-Numb-IRES-GFP), plated on methylcellulose, and colony numbers were
counted. For secondary plating, cells from primary plating were
harvested, replated, and colonies counted (n=3, p<0.05). While Numb
expression did not alter AML colony formation in the primary plating
(FIG. 5A) it led to 3-fold fewer colonies compared to control by the
secondary plating (FIG. 5B). Error bars in FIGS. 5A and 5B indicate
s.e.m.

[0089] To test the influence of Numb on the growth of established AML in
vivo, AML cells were isolated, infected with either vector or Numb, and
transplanted into irradiated mice. Survival was monitored over time and
compared. The incidence and latency of the disease in both groups were
similar in the primary transplant (FIG. 5C). However, when cells were
analyzed via FACS for frequency of lineage negative cells to determine
and compare the level of differentiation in the AML cells with and
without Numb, the leukemias differed in their cellular composition, with
Numb expressing leukemias showing a significant decrease in
undifferentiated lineage negative cells compared to control (36% vs. 63%
FIG. 5D, 5E; p=0.0496; error bars show s.e.m.). FIG. 5E shows the average
percentage of Lin- cells from primary transplanted mice. FIG. 5F shows
representative examples of cancer stem cells frequency
(Lin-Sca1+Flk2+CD150-) in primary leukemias. FIG. 5G shows the average
cancer stem cell frequency in primary leukemias (control vector, n=3 and
Numb, n=4; one statistical outlier from the Numb cohort was excluded
based on Grubb's test, p=0.045).

[0090] In addition, cells from primary transplanted mice were sorted for
donor derived cells, transplanted into irradiated recipients, and the
survival of these mice was monitored. As shown in FIG. 5H (data shown is
from three independent experiments; n=13 for Vector; n=15 for Numb;
p=0.0002), Numb had a significant impact on disease progression in
secondary transplants. While 78% ( 7/9) of mice transplanted with control
cells succumbed to leukemia, only 30% ( 3/10) of mice transplanted with
Numb expressing AML cells developed disease. Leukemic spleen cells from
secondary transplanted mice with AML/Vector (FIG. 5I) or AML/Numb (FIG.
5J) were cytospun and Wright stained, and it was observed from the
morphology that the leukemias that did develop in the presence of Numb
were significantly more differentiated (arrows indicate myeloblasts in
FIG. 5I and differentiated myeloid cells in FIG. 5J; magnification is
100×). These data indicated that continual Numb repression may be
important to AML establishment as well as maintenance even after the
disease is fully established. Importantly, leukemias that developed in
the presence of Numb were more differentiated (FIG. 4B, 4C), and unable
to propagate disease efficiently (93% versus 20%, FIG. 1g) or infiltrate
secondary organs (FIG. 4E, 4F, FIG. 6); no signs of leukemia were
detected in mice that survived (FIG. 4G, FIG. 6). Numb also impaired
propagation of fully established leukemias and dramatically reduced the
frequency of cancer stem cells (FIG. 5). Shown in FIG. 6 are leukemic
cells from mice transplanted with control- or Numb-expressing blast
crisis CML that were sorted, transplanted, and spleen sections obtained
and analyzed. Shown are hematoxylin and eosin staining of spleen sections
from a, leukemic vector infected transplants (FIG. 6A), leukemic Numb
infected transplants (disease at 40 days, FIG. 6B), and healthy Numb
infected transplants (sacrificed at 150 days, FIG. 6C). Original
magnification was at 63×, red arrows indicate areas of immature
myeloid cells, and black arrows indicate lymphoid follicles. These data
show that continual repression of Numb is essential for maintenance of
blast crisis CML, and that increasing the levels of Numb can inhibit
disease.

Example 5

Relationship of Numb and Notch in CML

[0091] It was tested whether Numb and Notch had a reciprocal relationship
in CML. Notch signaling was elevated in blast crisis CML (FIG. 7). Notch
signaling was differentially active in chronic and blast crisis CML. FIG.
7A shows a schematic of the strategy to detect Notch signaling in chronic
and blast crisis CML. KLS GFP+ cells from transgenic Notch reporter mice
(TNR) were sorted and infected with either BCR-ABL to generate chronic
phase disease or BCR-ABL and NUP98-HOXA9 to generate blast crisis CML.
Infected cells were transplanted into irradiated recipients (chronic
phase, n=3 and blast crisis, n=4). Following leukemia development, cells
were isolated from the bone marrow and analyzed by FACS for TNR reporter
activity as assessed by GFP expression. FIGS. 7B and 7C show GFP
expression (black line) in donor derived cells from (B) chronic and (C)
blast crisis CML. Leukemia cells from wild type mice were used as
negative control for GFP (gray line). FIGS. 7D and 7E show blast crisis
CML cells from TNR mice were sorted for GFP low and GFP high, cytospun
and immunostained with anti-cleaved Notch1 antibody (red) and DAPI
(blue). Presence of ICN was higher in GFP high cells confirming Notch
reporter activity correlated with cleaved ICN.

[0092] Notch signaling inhibition via dnXSu(H) delivery or through
conditional deletion of Rbpj paralleled the effects of Numb and led to
reduced incidence and propagation of blast crisis CML (FIG. 8) Inhibition
of Notch signaling lead to impaired development and propagation of blast
crisis CML. FIG. 8A shows data for blast crisis CML cells that were
infected with either vector control or dominant negative Xenopus
Suppressor of Hairless (dnXSu(H)), sorted, transplanted into irradiated
recipients, and survival monitored. Data shown is from two independent
experiments (Vector, n=10 and dnXSu(H), n=9, p=0.66). FIG. 8B shows data
for cells from primary transplanted mice that were sorted for
donor-derived cells, serially transplanted into irradiated recipients,
and survival monitored. Data shown is from two independent experiments
(n=10, **p<0.0001). FIG. 8C shows data for bone marrow progenitors
from control (+/+) mice or conditional Rbpj knockout (Rbpj-/-) mice that
were infected with NUP98-HOXA9 and BCR-ABL, transplanted into irradiated
recipients, and survival monitored. Data shown is from three independent
experiments (wild type, n=11 and Rbpj-/-, n=14, p=0.809). FIG. 8D shows
data for cells from primary transplanted mice that were sorted for
donor-derived cells, serially transplanted into irradiated recipients,
and survival monitored (Vector, n=5 and Rbpj-/-, n=4, *p=0.012).

[0093] In addition, levels of p53, another Numb target, were higher in
Numb-expressing blast crisis CML (FIG. 9A). In the absence of p53, Numb
was unable to impact leukemic cell growth in vivo or in vitro (FIG.
9B-F), indicating that Numb's effects are in part dependent on p53. Loss
of p53 impaired Numb's ability to inhibit blast crisis CML propagation.
FIG. 9A shows Lin- cells from Vector- or Numb-expressing
NUP98-HOXA9/BCR-ABL-induced leukemia that were sorted, cytospun, and
immunostained with anti-p53 (red) and DAPI (blue). FIG. 9B shows data for
bone marrow cells from p53 null mice (p53-/-) that were infected with
BCR-ABL and NUP98-HOXA9 together with either Vector or Numb, transplanted
into irradiated recipients, and survival monitored. Data shown are from
three independent experiments (Vector, n=10 and Numb, n=9, p=0.0149).
FIG. 9C shows data for donor-derived cells from primary transplants that
were re-transplanted and survival monitored for secondary disease. Data
shown are from two independent experiments (Vector, n=9 and Numb, n=10,
p=0.0918). FIG. 9D shows the frequency of the Lin- population from
primary and secondary leukemia. Error bars show s.e.m. For primary:
Vector, n=7 and Numb, n=5; secondary: Vector, n=7 and Numb, n=9. For FIG.
9E, cells from primary transplants were sorted, plated in methylcellulose
media, and colony numbers counted. Error bars show s.e.m. (n=2). For FIG.
9F, cells from primary plating were harvested and replated for secondary
colonies (*p=0.029). Data is representative of two independent
experiments. Error bars show s.e.m.

Example 6

Numb Expression is Diagnostic for Imatinib Non-Responsiveness

[0094] It was next tested whether Numb is differentially expressed in
human CML, imatinib resistant CML, and blast crisis CML (bcCML) cells in
human disease. CML cells were subdivided into two groups: CML with major
or complete molecular response (MMR or CMR), which can be easily cured
with Imatinib Mesylate (IM), and CML with no MMR, which may relapse after
Imatinib Mesylate (IM) treatment. The average BCR-ABL transcript of CML
with MMR or CMR was 0.033% on the International Scale (IS), whereas that
of CML with no MMR was 2.055% IS 12 months after IM treatment. To analyze
Numb expression in these samples, CML cells from bone marrow cells were
collected from human CML and bcCML patients before IM treatment. The bone
marrow were purified, RNA was isolated, and the level of Numb expression
was analyzed by realtime PCR with results normalized to actin expression
levels (n=5 for CML with MMR or CMR; p=0.07 and n=4 for CML with no MMR;
p=0.03 and n=0.03 for bcCML). Numb expression levels in CML with MMR or
CMR was higher compared to CML with no MMR and bcCML (FIG. 10). These
data suggest that Numb expression is downregulated as disease becomes
more aggressive, and Numb could be used as a prognostic marker for
imatinib non-responsiveness within CML patients.

Example 7

Musashi as a Therapeutic for Acute and Blast Crisis Myeloid Leukemia

[0095] The expression levels of the two paralogous mammalian Musashi
genes, Msi1 and Msi2, were examined using real time PCR. KLS cells were
sorted, and RNA was extracted and reverse transcribed. Following
equalization of template cDNA, Msi1 and Msi2 transcript expression was
analyzed by realtime PCR. It was found that Msi2 was dominant in normal
and transformed hematopoietic cells, while Msi1 was barely detectable in
these tissues.

[0096] Subsequent studies focused on Msi2. CML and AML cells were sorted
from spleen, RNA was extracted and reverse transcribed, and Msi2
expression levels were determined by realtime PCR with results normalized
to beta-2-microglobulin. Msi2 expression was 10-fold higher in AML than
in CML (FIG. 11A; CML, n=6; AML, n=9; error bars represent s.e.m.
p<0.001). AML cells were sorted into Lin.sup.- or Lin.sup.+ fractions
based on the expression of lineage markers, and Msi2 levels were
analyzed. As shown in FIG. 11B (n=5 for each group; p=0.039; error bars
represent s.e.m.), the expression of Msi2 was further enriched in the
lineage negative fraction within AML. These data indicated that Msi2
expression is associated with the most immature leukemic cells.

[0098] Since Msi2 and Numb were expressed in a reciprocal pattern, it was
examined whether Msi2 could downregulate Numb during leukemogenesis.
Isolated CML cells were infected with Msi2-expressing or empty vector.
Infected cells were sorted and stained with an anti-Numb antibody, and
nuclei were visualized by DAPI staining (pseudo-colored in green in FIG.
12F), to analyze expression of Numb by immunofluorescence (**p<0.001).
Fluorescence intensity of Numb staining was quantified by Metamorph
software, more than thirty cells from each cohort were analyzed,
background fluorescence intensity was subtracted from individual
fluorescence intensity, and averaged values are shown in FIGS. 12G and
12H (p<0.001). Msi2 significantly decreased the levels of Numb (FIG.
12F right panel and 12G) compared to empty vector control (FIG. 12F left
panel and 12G).

[0099] To determine whether the NUP98-HOXA9 oncoprotein itself plays a
role in how Msi2 expression is initially upregulated in AML, it was
tested if NUP98-HOXA9 can induce Msi2 expression. KLS cells were isolated
and transduced with BCR-ABL and vector, or BCR-ABL and NUP98-HOXA9.
Analysis of Msi2 expression with realtime PCR revealed that the presence
of NUP98-HOXA9 led to significantly higher levels of Msi2 (FIG. 12H);
data shown are from three independent experiments, *p=0.017), showing
that NUP98-HOXA9 could also activate this cascade by increasing
expression of Msi2. These data cumulatively suggest that Msi2 is
upregulated following NUP98-HOXA9 expression, and that Msi2, through its
ability to downregulate Numb, contributed to the establishment and
maintenance of acute myeloid leukemia and, at least in part, Msi2 has the
ability to activate a cascade that leads to inhibition of
differentiation.

[0100] Since NUP98-HOXA9 initiates transformation through HoxA9 mediated
DNA binding and transcription, it was tested whether HoxA9 could bind the
Msi2 promoter and activate its expression directly. As shown in FIG.
12I-L, HoxA9 bound to the Msi2 promoter. Shown in FIG. 12I is the murine
Msi2 gene structure: exons (numbered boxes), transcription start site
(TSS; +1) and the direction of transcription (flag), putative HOX binding
element 5.7 kb upstream of TSS (oval), and +110 kb site with no HoxA9
binding sequence (open rectangle). Chromatin immunoprecipitation (ChIP)
was performed either with IgG control or anti-HoxA9 antibody, wherein
Flt3, a known HoxA9 target gene, was used as a positive control (FIG.
12J). Msi2 -5.7 kb region (FIG. 12K) or Msi2 +110 kb region (FIG. 12L)
were also used as a positive control. The ChIP revealed that HoxA9 was in
fact associated with the putative HoxA9 binding element that was
identified at -5.7kb.

[0101] As shown in FIG. 12M, KLS cells from Msi2 genetrap reporter mice
were transduced with BCR-ABL with either control vector or NUP98-HOXA9
and (β-galactosidase reporter activity quantified (n=2 each,
*p=0.011). This indicated that NUP98-HOXA9 expression was also able to
induce Msi2 reporter activity in KLS cells. These data show that Msi2 can
be upregulated by NUP98-HOXA9 and subsequently contribute to blast crisis
CML by repressing Numb.

[0102] To test if Msi2 is required for the development of blast crisis
CML, a mouse in which the Msi2 gene was disrupted by a genetrap (Gt)
vector was utilized (Taniwaki, T. et al. Dev Growth Differ 2005, 47,
163-172, incorporated herein by reference in its entirety). FIG. 13A
shows a schematic illustration of a Msi2 mutant allele generated by an
insertion of a genetrap vector pU-21. Inverse PCR strategy allowed
identification of the location of the genetrap event in intron 5 of Msi2
gene. As genetrap vector pU-21 harbors splice acceptor sequence (SA) and
transcription termination/polyadenylation signal (pA) downstream of
2 -geo sequence, Msi2 gene transcription would be terminated at this
site. Shown in FIG. 13B are results from Realtime RT-PCR analysis of Msi2
expression in whole bone marrow cells from control (+/+) and homozygotes
for the genetrap allele (Gt/Gt) (n=6 each, **p<0.001). Results shown
were normalized to beta-2-microglobulin levels. Error bars represent
s.e.m.

[0103] Shown in FIG. 14B, is the frequency of KLS cells in mice of
indicated genotypes (+/+, n=4, +/Gt, n=3 and Gt/Gt, n=4). Shown in FIG.
14B is the survival curve of mice transplanted with BCR-ABL and
NUP98-HOXA9 infected +/+ or Gt/Gt KLS cells (+/+, n=15 and Gt/Gt, n=14,
*p=0.0159). Msi2 mutant mice were viable, albeit smaller and less
frequent than predicted (+/+:+/Gt:Gt/Gt=38:66:19, p=0.038), and showed a
two-three fold reduction in the frequency (FIG. 14A) and absolute numbers
(data not shown) of KLS cells. Additionally, the loss of Msi2 led to
significantly impaired leukemia growth in vivo (FIG. 14B, 93% for control
versus 57% for Gt/Gt).

[0104] To determine if inhibiting Msi2 could impact the growth of
established CML, and to rule out the possibility that the reduced
incidence of leukemia in Gt mutants was due to developmental defects and
examine if Msi2 knockdown affects AML maintenance, Msi expression was
targeted using an alternate shRNA approach. A lineage-negative population
was sorted from full blown AML cells, which contains AML stem cells. Lin-
cells from NUP98-HOXA9/BCR-ABL-induced blast crisis leukemias were
infected with either an shRNA retrovirus targeting Msi2 (shMsi; SEQ ID
NO: 9) or firefly luciferase (shLuc; SEQ ID NO: 10) as a negative control
and resorted, and Msi2 expression was analyzed by realtime RT-PCR.
Expression levels were normalized to the level of beta-2 microglobulin
and displayed relative to the control arbitrarily set at 100. Error bars
represent s.e.m. of triplicate PCRs (**p<0.01). Results are shown in
FIG. 15A. Knockdown of Msi2 resulted in 75-90% reduction in Msi2
transcript levels compared with the shLuc control. A colony forming assay
was done using methylcellulose media, and it was found that Msi2
knockdown led to 50-70% reduction in the number of colonies formed,
suggesting that Msi2 reduction impaired colony forming ability of the AML
cells.

[0106] Delivery of Msi2 shRNAs (shMsi) into established blast crisis CML
cells also reduced disease incidence in vivo, as shown in the survival
curve of mice transplanted with established blast crisis CML cells
infected with control shLuc or shMsi (FIG. 14D, n=13 each, *p=0.0267).

[0107] Further, the majority of leukemias that occurred in the presence of
shMsi were more differentiated (FIG. 14E), and impaired in their ability
to propagate disease (FIG. 14F, 87% control versus 25% shMsi). FIG. 14E
shows Wright's stain of leukemic cells from mice transplanted with
control shLuc or shMsi infected blast crisis CML, with immature
myeloblasts (closed arrowheads) and differentiating myelocytes and mature
band cells (open arrowheads) indicated (magnification: 100×). FIG.
14F shows the survival curve of mice transplanted with Lin.sup.- cells
from primary shRNA expressing leukemias (control, n=15 and shMsi, n=16,
**p<0.001). Data shown is representative of two to three independent
experiments. These data show that Msi2 is important for establishment and
continued propagation of blast crisis CML.

[0108] The impact of Msi2 loss in AML propagation in vivo was also tested.
Lin- negative cells from full blown AML were transduced either with an
Msi2 shRNA retrovirus or luciferase shRNA virus, and the cells were
transplanted into sublethally-irradiated mice. 11 of 13 mice transplanted
with control knockdown AML became sick by 1 month post transplant. In
contrast, more than half of the mice transplanted with Msi2 knockdown AML
survived beyond 1 month (FIG. 16). The surviving mice had no detectable
levels of transplanted leukemic cells in their peripheral blood,
indicating loss of the leukemic cells.

[0109] Serial transplants were also done to test the presence of
leukemia-initiating ability of control and Msi2 knockdown AML cells from
the mice who suffered from leukemia. While control knockdown AML cells
were able to propagate disease in 2 out of 3 mice (transplanted with 3000
cells) or all of the mice (4 out of 4 at 10,000 cells transplanted),
Msi2-knockdown AML cells were significantly impaired in their ability to
initiate AML leading to a loss in AML incidence at either of the cell
doses used. These data suggested that AML required Msi2 to be highly
expressed during disease maintenance and propagation.

Example 8

Msi2 in Human Leukemia Progression

[0110] To determine if human myeloid leukemias have similar disease
progression, the expression levels of Musashi (MSI2) were analyzed in
human patient specimens from chronic phase and blast crisis CML patients.
9 specimens were obtained for each of chronic phase (CP) and blast crisis
(BC) CML, and cDNA were prepared from the total RNA from these specimens.
By using realtime RT-PCR analyses, it was found that the human ortholog
MSI2 transcript levels were 6-fold higher in BC CML than in CP CML (FIG.
17) suggesting that a similar mechanism upregulating Musashi operates in
both mice and humans to drive blast crisis CML.

[0111] Finally, it was examined if MSI2 was aberrantly upregulated during
human leukemia progression. MSI2 was tracked in 30 patient samples from
banks in Korea and the United Kingdom, and found to be expressed at
significantly higher levels in blast crisis CML (FIG. 18A, 18B). FIG. 18A
shows data from PCR analysis of MSI2 expression in chronic and blast
crisis CML patient samples from the Korean Leukemia Bank (n=9 per cohort,
Mann-Whitney U test **p<0.001) and FIG. 18B shows data from the
Hammersmith MRD Lab Sample Archive in the United Kingdom (n=6 per cohort,
Mann-Whitney U test, **p<0.001).

[0112] To determine if this reflected a general pattern in human CML
progression, the expression of MSI2 and associated genes were examined in
90 patient samples from banks in the United States (Radich, J. P. et al.
Proc Natl Acad Sci USA 2006, 103, 2794-2799, incorporated herein by
reference in its entirety). FIG. 18 shows data from microarray analysis
of expression of MSI2 (FIG. 18C, p<0.001), NUMB (FIG. 18D,
p<0.001), HOXA9 (FIG. 18E, p<0.001), and HES1 (FIG. 18F, p=0.68) in
bone marrow and peripheral blood samples from 42 chronic (red), 17
accelerated (green), and 31 blast crisis phase (blue) patients in the
United States. Microarray analysis revealed a dramatic upregulation of
MSI2 in every patient during CML progression (FIG. 18C). In addition,
NUMB was downregulated in a majority of blast crisis patients (FIG. 18D).
Notably, our mouse model was driven by NUP98-HOXA9 as a second hit,
whereas human blast crisis CMLs harbor a variety of secondary mutations.
Msi2 could be regulated by HoxA9 expression in the mouse model of CML,
and it was examined if HOXA9 was upregulated in blast crisis CML samples.
The observation that a majority of patient samples had elevated levels of
HOXA9 (FIG. 18E) may explain how MSI2 becomes upregulated in advanced
stage disease regardless of the nature of the second hit. Notch signaling
targets HES1 and TRIB2 were also elevated in a number of blast crisis
patient samples (FIG. 18F, FIG. 19). Notch signaling-associated genes
HES1 and TRIB2 are elevated in blast crisis patient samples. FIG. 19A
shows HES 1 expression during CML progression within a single patient,
determined via PCR analysis of HES1 in sorted CD34+ chronic phase and
blast crisis phase cells from the same individual (Singapore General
Hospital). Error bars represent s.e.m. of triplicate PCRs. FIGS. 19B and
19C show results for chronic and blast crisis phase cells from CML
patient samples (United Kingdom and Korean banks) that were isolated, RNA
prepared, and TRIB2 expression analyzed by realtime RTPCR (FIG. 19B,
expression levels in individual samples; FIG. 19C, average expression
levels n=11 for each leukemia, Mann-Whitney U test, *p=0.0031).

[0113] Because the highest MSI2 expression was observed in blast crisis
patients, where treatment outcomes are extremely poor, and because a
range of expression was observed in both chronic and accelerated phase
CML, it was tested whether MSI2 expression correlated with outcomes after
allogeneic transplantation. Patients were divided into two groups based
on median expression of MSI2. Among 37 chronic phase patients with
available outcomes (9 relapses) increased MSI2 expression was associated
with a higher risk of relapse (hazard ratio=4.35; 95% CI, 0.90-21.06,
p=0.07). Additionally, among 13 accelerated phase patients with available
outcomes (6 deaths and 3 relapses), increased MSI2 expression was not
only associated with higher risk of relapse (all relapses occurred in the
increased MSI2 group, p=0.06), but also with higher risk of death (hazard
ratio=6.76; 95% CI, 0.78-58.57, p=0.08). The association of MSI2 with
poorer outcomes suggests that Msi2 may be an early marker of advanced CML
disease.

[0114] Our work identifies the Musashi-Numb axis as an important regulator
of myeloid leukemia and indicates that maintenance of the immature state
is dependent on reversal of classic differentiation cues. Specifically,
it was found that Msi2 is upregulated and Numb downregulated as chronic
phase CML progresses to blast crisis, and that modulation of this pathway
can inhibit disease. Without being limited as to theory, a proposed model
for the role of Musashi and Numb in CML progression is shown in FIG. 18G.
The Musashi and Numb pathway is required for hematologic malignancy.

[0115] Numb, which drives commitment and differentiation, can impair blast
crisis CML establishment and propagation. Just as Numb's influence may be
mediated through p53 and/or Notch signaling, Musashi may act through Numb
as well as other targets such as p21WAF1. Specific differentiation cues
associated with the Musashi-Numb cascade may unlock the differentiation
potential of blast crisis CML and impair its growth. Musashi may be an
early marker of advanced CML. Musashi expression may serve as a
prognostic tool, and targeting it may be used in therapy.

[0116] Various features and advantages of the invention are set forth in
the following claims.